The development of antibiotic resistance mechanisms hinders the treatment process. So far, there is limited data on the problem of bacterial resistance in hospitals in Central and Eastern Europe. Therefore, this study aimed to assess the prevalence of resistance mechanisms and alert pathogens based on reports regarding cultures of samples collected from general hospital patients in Poland in the period 2019–2021. This study examined the prevalence of resistance mechanisms and alert pathogens and the structure of microorganisms, including the type of diagnostic material in the hospital department. The frequency of occurrence and the trends were analysed based on Cochran’s Q-test, relative change and the average annual rate of change (AARC). Of all 14,471 cultures, 3875 were positive for bacteria, and 737 were characterised by resistance mechanisms (19.0%). Alert pathogens were identified in 983 cases (24.6%), including pathogens isolated from blood samples. The most commonlyisolated bacteria were Escherichia coli (>20% of positive cultures), Enterococcus faecalis (7%), and Klebsiella pneumoniae (6%) increasing over time; Staphylococcus aureus (13%) was also found, but its proportion was decreasing over time. Extended-spectrum β-lactamase (ESBL) was the most frequent resistance mechanism in Internal Medicine (IM) (p < 0.001) and the Intensive Care Unit (ICU) (p < 0.01), as well as in ICU-COVID; this increased over the study period (AARC ↑34.9%). Methicillin-resistant Staphylococcus aureus (MRSA) (AARC ↓50.82%) and AmpC beta-lactamase (AARC ↓24.77%) prevalence fell over time. Also, the number of alert pathogens was dominant in the IM (p < 0.01) and ICU (p < 0.001). The most common alert pathogen was ESBL-positive E. coli. Gram-negative rods constitute a significant epidemiological problem in hospitals, especially the growing trend of ESBL in IM and ICU, which calls for increased control of sanitary procedures.