In Oman, mastitis is an important disease that affects the dairy animals. This study was conducted to identify the major bacterial and mycotic pathogens associated with bovine mastitis cases in Oman. For isolated bacteria, two virulence factors, antibiotic profiles, different isolates relatedness, seasonality and age effect were investigated. Between June 2017- January 2018, a total of 76 quarter milk samples were collected from 30 cows with clinical and subclinical mastitis from different governorates including; Dakhiliyah (54%), Ad Dhahirah (20%), Al Batinah (16%), Ash Sharqiyah north (5%) and Muscat (3%). Different physical and chemical test were carried out for milk samples; color, consistency, modified wide side tests (MWT) and pH. The bacteria and fungi were identified using using 16S rDNA and 18S rDNA genes, respectively. Sequencing and VITEK 2 system results were consistent for only 40% of the bacterial isolates at the species level. Samples with subclinical mastitis (57, 75%) were higher than with clinical mastitis (19, 25%). Bacterial isolates were detected in 82% of the samples. Mixed infections were detected in 12% of the milk samples and 18% of the samples were culture negative for bacteria. The majority of mastitis cases were associated with environmental pathogens. The most predominant isolated bacteria were coagulase negative staphylococci (CNS) in clinical and subclinical mastitis cases. Nine of the identified environmental pathogens namley Achromobacter insolitus, Bacillus velezensis, Bacillus australimaris, Bacillus xiamenensis, Bacillus aryabhattai, Brevibacillus borstelensis, Brevibacillus agri, Cosenzaea myxofaciens, Providencia rettgeri were reported here for the first time in the world to be associated with mastitis. Hemolysis activity was detected in 79 % of the strains while coagulase enzyme activity was detected in 42 %. Antibiotics profiles showed that the bacterial isolates have differential sensitivities to seven tested antibiotics, which are commonly used. Majority of isolates were resistance to Ampicillin and many were multidrug resistance. About half of the examined samples (47%) were positive for fungal growth. Candida yeast was the most predominate isolates (66%) followed by Penicillium (28%), Aspergillus spp (16%) and Galactomyces geotrichum (12%). Most of the environmental pathogens were isolated in summer while contagious pathogens were more frequent in winter (P<0.05). Cows in the age group 5-8 years were more affected in this study. Molecular identification not only allowed to identify microbes accurately at the species level, but showed the presence of different haplotypes associated with mastitis in Oman. Also, suggested that the overuse of antibiotics in treatment of mastitis might be implicated in the emergence of multi antibiotic resistant bacteria. The presence of mycotic and environmental mastitis suggested that unhygienic practices in the farm and improper management are potentially major contributors for mastitis.