•5.4% and 1.1% prevalence of MSSA and MRSA respectively.•19 different spa types were detected including a novel type (t16571).•Low risk perception and poor hygiene practices employed by abattoir workers in the study areas.•Formal education and occupation were associated with S. aureus knowledge levels.•Several risk factors predispose abattoir workers to bacterial colonization. Staphylococcus aureus is a zoonotic pathogen of significant public health concern. Information on the prevalence and risk factors facilitating bacterial colonization and spread under abattoir settings in Nigeria are scarce. This cross-sectional study was designed to determine prevalence of S. aureus as well as risk factors on knowledge and practices facilitating pathogen carriage among workers and slaughter animals in two municipal abattoirs of Ilorin and Ibadan, Nigeria. Swab samples (n = 1671) from nostrils of cattle, goats, pigs and abattoir workers, and from meat tables and abattoir walls were collected for detection of S. aureus. A questionnaire was administered to 275 workers to elucidate risk factors of pathogen carriage applying a logistic regression model. S. aureus prevalence was 6.5%. In total, MSSA and MRSA were detected at a frequency of 5.4% and 1.1%. Molecular analysis of the isolates revealed 19 different spa types, including a novel spa type (t16751). Gender, marital status, occupation and abattoir location were factors influencing worker’s practices in relation to pathogen carriage and spread in the abattoir setting. This present study detected not only low MSSA and MRSA prevalence, in both abattoirs but also low risk perception and hygiene practices employed by abattoir workers. Good practices among workers at Nigerian abattoirs are needed to mitigate S. aureus carriage. Further studies expounding the antibiotic resistance and relationships of MSSA and MRSA strains detected in this study are needed to complement understanding of the spread of S. aureus in the abattoir food chain.