This study was designed to determine the prevalence and co-infection of Mycobacterium tuberculosis and other pathogenic bacteria among presumptive cases of tuberculosis (TB) at selected hospitals in Mwanza, Tanzania. GeneXpert and conventional bacteriological culture and sensitivity were used for the detection of TB and other pathogenic bacteria, respectively. STATA version 13.0 was used for data analysis. The median (IQR) age of participants was 33 (19–51) years with males forming more than half (i.e., 59% (158/264)) of the participants. Microscopically, 29.5% (78/264) of the patients had polymorphonuclear leucocytes in the sputum samples. Approximately 7.2% (19/264), 16.3% (43/264), and 1.1% (3/264) of participants had TB, other pathogenic bacteria, and co-infections, respectively. One sample had growth of two other bacteria, resulting in a total of 44 isolated bacteria with the predominance of Gram-negative bacteria at 75.0% (33/44). The predominant species isolated was the Klebsiella pneumoniae complex at 52.3% (23/44). Overall, 27.3% (9/33) of GNB were resistant to third-generation cephalosporins, while Gram-positive bacteria were more resistant to erythromycin at 63.6% (7/11). Good quality sputa had a significantly higher yield of pathogenic bacteria than poor quality sputa (37.2% vs. 7.5%, p < 0.001). Presumptive TB cases were predominantly infected with other pathogenic bacteria than M. tuberculosis. Therefore, other pathogenic bacteria should be considered when attending presumptive TB cases to ensure favorable treatment outcomes.