Background: The toxic shock syndrome toxin (TSST-1) is important in the pathology of toxic shock syndrome. However, little data exist on its prevalence among clinical isolates of S. aureus in Nigeria. Hence, this study was carried out to detect the tsst-1 gene and associate it with phenotypic antibiotic resistance in clinical isolates of S. aureus. Methods: Staphylococcus aureus isolates were presumptively identified by Gram's staining and conventional biochemical tests while confirmatory identification was through the detection of the thermonuclease (nuc) gene. Antibiotic sensitivity testing was carried out using the modified Kirby-Bauer disc diffusion method while phenotypic detection of methicillin resistance was carried out using the cefoxitin disc sensitivity assay. The tst gene was detected within the genome of the bacterial isolates using Uniplex polymerase chain reaction (PCR). Results: Of the 152 S. aureus isolates identified in this study, 103 (67.76%) encoded the tst gene. Of these 103 tst-positive isolates, 63 (61.16%) were methicillin-resistant while 40 (38.84%) were methicillin-sensitive. The tst-positive isolates (n=103) were resistant to tetracycline (39.81%), erythromycin (24.27%), gentamicin (22.33%), cotrimoxazole (22.33%), ciprofloxacin (21.36%), fusidic acid (16.5%), fosfomycin (10.68%), and clindamycin (5.82%). Comparatively, tst-negative isolates (n=49) were resistant to tetracycline (69.39%), cotrimoxazole (56.06%), gentamicin (53.06%), ciprofloxacin (51.02%), erythromycin (46.94%), fusidic acid (28.57%), fosfomycin (26.53%), and clindamycin (8.16%). Phenotypic antibiotic resistance is significantly associated with the presence of the tst gene (p < /em><0.05) except for clindamycin and fusidic acid (p < /em>>0.05). Coclusion: Hence, the high prevalence of the tst gene and its association with antibiotic resistance in S. aureus is a cause for worry.