Hepatotoxicity is reported frequently as an adverse reaction during tuberculosis (TB) and HIV treatment. This study aimed to investigate the incidence of antiretroviral and anti-tuberculosis druginduced liver enzymes activities variation in TB and TB-HIV co-infected patients at Jamot Hospital in Yaoundé-Cameroon. From April 2018 to May 2019, 336 treatment-naïve TB patients were enrolled. Liver enzymes (AST, ALT, ALP) and total bilirubin were evaluated at baseline and 12 weeks after treatment initiation. Blood was spotted on filter paper for DNA extraction by the chelex method. Standard nested PCR followed by restriction enzyme analysis with KpnI, TaqI, and BamHI to detect NAT2 polymorphisms was performed. TB-HIV co-infection prevalence was 29.46%. There was a significant rise of transaminases (p < 0.05) at baseline in TB-HIV co-infected patients. At 12 weeks, there was a substantial rise of transaminases in TB patients, and total bilirubin in TB-HIV co-infected patients (p < 0.05). The prevalence of slow and fast acetylators was 85.71 and 14.29%, respectively. NAT2*5/5 and NAT2*5/6 genotypes were most represented. Slow acetylating NAT2 phenotype was significantly associated with drug hepatotoxicity (p < 0.05). The prevalence of TB-HIV co-infections remains high, and the rise in transaminases is linked to the slow acetylating NAT2 phenotype.