Lactobacillaceae is a large family of bacteria from which probiotic strains often originate. Microorganisms used as feed additives in the EU must meet a number of formal criteria, some of which concern antimicrobial susceptibility. In this study, we determined the susceptibility of 19 reference strains and 121 wild-type strains of Lactobacillaceae to aminoglycoside antibiotics using the broth microdilution method based on the ISO 10932:2010/IDF 223:2010 standard. Strains were categorized as resistant or susceptible according to European Food Safety Authority (EFSA) guidelines. Resistance genes were detected by whole genome sequence (WGS) analysis or by PCR. The MICs read after 48 h of incubation showed that 36.8% of reference strains were resistant to kanamycin, 26.3% to streptomycin, and 5.3% to gentamicin, with no aminoglycoside resistance genes detected in any genome. As many as 93.2% of field isolates of Ligilactobacillus salivarius, 85% of Ligilactobacillus agilis, and 58.8% of Lactiplantibacillus plantarum were classified as resistant to kanamycin, with the aac(6)-Ie-aph(2)-Ia gene detected only in two isolates. In six of 12 streptomycin-resistant strains, the ant(6)-Ia gene was identified, which usually coexisted with the spw gene. Three isolates with high neomycin MICs harbored the ant(4′)-Ia gene. In Lactobacillus gallinarum strain LMG 9435, characterized by streptomycin MIC value > 1024 µg/mL, a potential resistance-causing mutation in the rpsL gene (Lys56 → Arg) was detected. The results of the study indicate that some genera of Lactobacillaceae, in particular L. salivarius and L. agilis, exhibit natural resistance to aminoglycoside antibiotics, mainly kanamycin. Therefore, there is a need to update the EFSA guidelines on antimicrobial susceptibility testing of Lactobacillaceae, so that strains lacking resistance genes and/or chromosomal mutations are not considered to be resistant.