Responses to antitubercular drugs like isoniazid (INH) are influenced by genetic polymorphisms in metabolizing enzymes and transporters. This study is aimed at analyzing genetic polymorphisms of NAT2, CYP2E1, and GSTM1 genes in Saudi TB patients, monitoring INH drug levels, and exploring correlations between these genetic variations, drug levels, hepatotoxicity incidence, and clinical outcomes. This prospective cohort design was conducted at King Abdul-Aziz University Hospital in Jeddah, Saudi Arabia. It followed 50 TB patients undergoing first-line anti-TB treatment for 6 months. Genotyping and INH serum concentration measurements were conducted. The mean INH plasma drug levels measured in 30 patients were 2.86 ± 2.80. The presence or absence of the GSTM1 does not statistically affect the plasma INH level between the TB patients with no significant association between GSTM1 and clinical response, while high plasma concentration of INH was significantly associated with improved clinical response. The present study demonstrated no NAT2 and CYP2E1 gene variations in Saudi TB patients but has identified a GSTM1 variant in 68% of patients. The presence or absence of the GSTM1 gene variant appears to not affect INH drug level or clinical outcomes. Clinicians should consider individualized TB treatment based on genetic and demographic factors.