Staphylococcus species, mainly S. aureus, S. intermedius, and S. hyicus species, are the primary causes of intra-mammary infections in cows and causes human food poisoning infections; hence, this study aimed to assess the prevalence, antibiogram, virulence, and methicillin resistance gene profiles of pathogenic Staphylococcus species from bovine milk samples. A cross-sectional study design was employed to collect milk samples from February to August 2022 from 290 lactating cows in 38 small and medium-scale dairy farms. Isolation of pathogenic Staphylococcus species was performed by plate culturing and biochemical tests and the antimicrobial susceptibility profiles were determined by Kirby-Bauer disc diffusion method. Virulence and methicillin-resistant genes were detected using conventional PCR. Logistic regression and Chi-square test were used for data analysis. The overall Staphylococcus species prevalence was 16.6 % (95 % CI = 12.5-21.3). Of these, 52.1 %, 33.3 %, and 14.6 % were S. aureus, S. intermedius, and S. hyicus species, respectively. Farm management, herd size, parity, milk yield and breed risk factors had a significant association with the occurrence of Staphylococcus species. All the isolates showed 100 % resistance to amoxicillin and ampicillin discs, while 41.7 % (95 % CI = 27.6-56.8) of the isolates showed multidrug resistance. The study revealed that 68.8 %,33/48 (95 % CI = 53.7-81.3) of the pathogenic Staphylococcus isolates carried one or more of the virulence and/or methicillin resistance genes. The mecA, hlb, hla, icaD, pvl, tsst-1, and sec genes were detected in 58.3 %, 54.2 %, 50 %, 41.7 %, 29.2 %, 10.4 %, and 4.2 %, respectively, from 48 Staphylococcus isolates. The study revealed that pathogenic Staphylococcus isolates carried various virulence and became multidrug resistant; which necessitates employing Staphylococcus caused mastitis control and prevention measures.