The present study describes the probiotic potential and functional properties of the lactic acid bacterium Pediococcus pentosaceus OBK05 isolated from buttermilk. The isolate OBK05 was assessed for its probiotic properties. The isolate showed notable tolerance to pH 2.0 and 3.0 (8.44, 8.35 log CFU/mL), oxbile of 0.5% at 2 and 4 h of incubation (6.97, 6.35 log CFU/mL) and higher aggregation (auto-aggregation, adhesion to hydrocarbons) than the referral strain, Lactobacillus acidophilus MTCC 10307. The adhesion efficiency to HT-29 cells was found to be maximum, corresponding to 93.5% and 97% at 1 and 2 h incubation, respectively. In addition, the isolate OBK05 showed antagonistic solid activity against bacterial pathogens like Pseudomonas aeruginosa MTCC 424 and Bacillus subtilis MTCC 1133. The phenotypic antibiotic resistance of the isolate was examined before and after curing plasmids. Among the known five structural genes responsible for different antibiotic resistance, four genes indicating antibiotic resistance to kanamycin-Aph (3´´)-III, streptomycin-strA, vancomycin-vanA and ciprofloxacin-gyrA were detected by PCR amplification of genomic DNA. Further, the horizontal gene transfer from OBK05 isolate to pathogens was not found for these antibiotic resistance markers when filter and food mating were carried out as no transconjugants developed on media plates containing respective antibiotics. This indicates that the intrinsic resistance is harbored on chromosomal genes, and hence it is nontransferable to other microbes. In addition, strain OBK05 exhibited good DPPH scavenging activity of 56 to 77% and liberated free amino acid from conjugated bile acid. The strain OBK05 demonstrated a strong ability to reduce cholesterol at 12 h (17%), 24 h (27%) and 48 h (67%) of incubation.