The presence of bacteria in indoor air poses significant health and environmental risks, particularly in densely populated settings such as educational institutions. Accurate identification and quantification of indoor microbial populations are crucial for assessing health hazards and establishing air quality standards. This study investigated the bacterial load in classrooms at Rivers State University using the Koch sedimentation method. Additionally, the antibiotic susceptibility of bacterial isolates was evaluated via the Kirby-Bauer disc diffusion technique. Results revealed that during morning and peak class hours, the Faculty of Law classroom recorded the highest aerobic bacterial load (1.18 × 10⁴ CFU/m³), followed by the Chemistry classroom (9.44 × 10³ CFU/m³), while the Psychology classroom exhibited the lowest bacterial load (1.28 × 10³ CFU/m³). In the evening, after class activities, the Chemistry classroom had the highest bacterial count (4.90 × 10³ CFU/m³), with the Business Education classroom next (3.10 × 10³ CFU/m³), and the Psychology classroom again showing the lowest (9.17 × 10² CFU/m³). Statistically significant differences (P ≤ 0.05) were observed in bacterial loads, with the Chemistry laboratory showing the highest heterotrophic bacterial counts. The predominant bacterial genera isolated included Bacillus, Staphylococcus, and Micrococcus. Antibiotic susceptibility testing revealed that Staphylococcus isolates were highly resistant to Ampiclox but sensitive to Gentamycin, Chloramphenicol, and Ciprofloxacin. Bacillus species showed high susceptibility to Ampiclox, Rifampicin, Ciprofloxacin, and Erythromycin. Micrococcus isolates exhibited no resistance to any tested antibiotics. These findings highlight the importance of monitoring indoor bacterial contamination in educational settings to mitigate health risks and guide effective antimicrobial interventions.