Parcel vending machines (PVMs) are receiving more recognition as an environmentally friendly last-mile delivery service. However, their high popularity creates the risk of microbial contamination of touchscreens and keypads resulting in the spread of pathogens in humans. The study aimed at assessing the degree of bacterial contamination of PVMs and characterizing the microbial population using mass spectrometry-based identification. In total, 64 PVMs located in 16 districts of city Lublin (Poland) were studied for the total number of aerobic bacteria (TNAB) using contact plates, whereas bacterial identification was conducted using the MALDI-TOF MS. Study revealed that the average TNAB for the analyzed districts ranged from 1 ± 0.4 CFU/cm2 to 8.54 ± 10.77 CFU/cm2. Statistical analysis showed a positive correlation between the TNAB and the population density (p = 0.0193), emphasizing the influence of human microbiota on the level of bacterial contamination. Among 140 reliably identified bacterial species (96.3%), the most prevalent were Bacillus spp. (52.8%) and Staphylococcus spp. (10.7%). Multiple opportunistic pathogens were detected, including B. cereus and Escherichia coli. This study indicates the need to develop procedures for disinfection or to implement modified materials for the PVMs touchscreens to limit the adhesion of potentially pathogenic microorganisms.