Inanimate surfaces are often referred to as nosocomial bacterial reservoirs and represent an important vector in the process of spreading pathogens to patients. Most gram-negative rods can survive on inanimate surfaces for several months. The aim of this study is to determine the prevalence and resistance of gram-negative bacteria isolated from the inanimate surfaces of two selected hospital departments. MALDI-TOF identified gram-negative rods isolated from inanimate surfaces. Antibiotic resistance was determined using a disk diffusion method, and the phenotype of resistance was determined using an inhibitory analyzer. From the inanimate surfaces, 98 strains of gram-negative nosocomial bacteria were identified by the MALDI-TOF MS. The most frequently isolated bacterium occurring in both departments was Pseudomonas aeruginosa (n = 33), followed by Acinetobacter baumannii (n = 20) and Enterobacter cloacae (n = 14). The most common phenotypic type of resistance in both departments was ampicillin resistance—AmpC (n = 38), then production of extended-spectrum β-lactamase (ESBL) (n = 33), followed by SHV-1 (n = 11), TEM-1 (n = 11), and fluoroquinolone resistance—Qnr (n = 22). The nosocomial important enzymes capable of hydrolyzing carbapenems, OXA-48 and metallo-β-lactamases, were confirmed in 12 and 2 cases, respectively. The results of our study prove that inanimate surfaces in hospitals are a reservoir of resistant gram-negative bacteria, which directly threaten hospitalized patients.