Abstract Aims: Clinical feature and microbiological investigation help in reaching the diagnosis in cases of microbial keratitis. But in cases where a patient is already on polytopical therapy, clinical features are altered so dependency on microbiological investigation is more by the time cases reached to the referred hospital. The present study aimed to overview the profile of microbial investigation in microbial corneal ulcer cases that were already on polytherapy referred to the ophthalmic department of tertiary care center. Materials and Methods: This is a retrospective hospital record-based study where microbiological reports of 58 infective keratitis cases of moderate-to-severe nature were analyzed. Statistical analysis was calculated with confidence intervals (CIs) using “Medcalc” statistical software online. Results: Following are the results of the study: 52% patients were males; 18.97% cases were positive for Gram stain. Aerobic culture was positive in 10.34%; potassium hydroxide (KOH) mount was positive in 9.62% cases; sabouraud dextrose agar culture was positive in 7.69% cases. Sensitivity of Gram stain was 33.3% (95% CI: 4.33%–77.72%), whereas specificity was 82.7% (95% CI: 69.67%–91.77%); positive predictive value (PPV) was 18.18% (95% CI: 2.28%–51.78%) and negative predictive value (NPV) was 91.49% (95% CI: 79.62%–97.63%). Sensitivity of KOH mount was 25% (95% CI: 0.63%–80.59%), whereas specificity was 91.67% (95% CI: 80.02%–97.68%); PPV was 20.20% (95% CI: 0.51%–71.64%) and NPV was 93.62% (95% CI: 82.46%–98.66%). Pseudomonas aeruginosa and Staphylococcus aureus were the most common bacterial isolates. Conclusions: Basic microbiological investigations have a low yield in chronic infectious keratitis cases that are on prior cocktail long-term therapy. Therefore, advanced tests such as polymerase chain reaction, confocal microscopy, and corneal biopsy may be helpful in managing such challenging cases.