The present study collected bacterial samples from water and bottom sediments from fish farms located in a nature reserve area in Poland with no recorded history of antibiotic use. The aim of the study was to determine the initial states of tetracycline, streptomycin, and erythromycin resistance before a potential increase of intensive aquaculture and application of antimicrobial agents in that region. With this in mind, the diversity and antibiotic resistance phenotypes and genotypes of isolates from the bottom sediments and water in five of the 13 fish ponds in Raszyn were evaluated. A total of 58 (sediment, n = 24; water, n = 34) non-repetitive and non-susceptible isolates were affiliated to 14 genera. Among the sediment isolates, Pseudomonas spp. and Bacillus spp. were isolated most frequently, and from the water, Stenotrophomonas spp. and Pseudomonas spp. Phenotypically resistant isolates selected by disk diffusion were further screened by polymerase chain reaction (PCR) and amplicon sequencing. The isolates derived from the water showed a greater percentage of phenotypically resistant isolates to each of the three antibiotics. The most common tetracycline resistant genes detected in isolates from both the water and sediment were tet(A), tet(T), tet(W), and tet(34). On the other hand, the genes tet(X), tet(H), tet(M), and tet(BP) were the most frequent among sedimentary isolates, while tet(B), tet(C), tet(D), and tet(32) were prevalent in aquatic isolates. The most prevalent streptomycin resistance genes among the aquatic isolates were aac(6’)-I, str(A), and str(B). The erythromycin resistance genes detected in all isolates included msr(A), erm(X), erm(V), erm(F), and erm(E).