During the COVID-19 pandemic, changes occurred within the surgical patient population. An increase in the frequency of resistant Gram-negative bacteria has since been recorded worldwide. After the start of the COVID-19 pandemic, microbiological diagnostics in our institution was performed using MALDI-TOF mass spectrometry. With this study, we wanted to confirm whether it contributed to a greater number of pathogenic bacteria detected in surgical ICU patients. A total of 15,033 samples taken from 1781 surgical patients were compared during the period from 2016 to February 2020 and during the COVID-19 pandemic from March 2020 to February 2023. On patients’ admission, pathogenic bacteria were mostly isolated from the respiratory system (43.1% and 44.9%), followed by urine cultures (18.4 vs. 15.4%) before and during the pandemic. After the onset of the COVID-19 pandemic, there was a significant increase in the frequency of isolation of Enterobacter spp. (5.4 before vs. 9%, p = 0.014) and other enterobacteria (6.9 vs. 10.8%, p = 0.017) on patients’ admission to the ICU, respectively. Despite this change, mortality in the ICU during the post-COVID-19 period was reduced from 23 to 9.6% (p < 0.001). The frequency of bacterial isolation did not change with the application of MALDI-TOF technology. By identifying the microorganism while simultaneously recognizing some resistance genes, we were able to start targeted therapy earlier. With the application of other infection control methods, MALDI-TOF may have contributed to the reduction in mortality in surgical ICU patients during the COVID-19 pandemic.