Environmental enterococci are increasingly recognised as major reservoirs of antimicrobial resistance (AMR) determinants and reliable indicators of faecal contamination in aquatic and terrestrial ecosystems. This exploratory pilot study, conducted on a limited convenience sample from the Oran region (northwestern Algeria), pursued two interconnected objectives within a One Health framework: (i) to characterise the antimicrobial resistance profiles of environmental Enterococcus isolates recovered from hospital wastewater, municipal sewage sludge, manure-amended agricultural soil, and contaminated river water through species-specific sodA-targeted PCR; and (ii) to evaluate the preliminary in vitro antagonistic potential of four Lactobacillus spp. strains, confirmed at genus level by genus-specific PCR and pre-screened for safety, against multidrug-resistant (MDR) enterococcal isolates. A total of 13 isolates (8 Enterococcus faecalis, 5 Enterococcus faecium) were confirmed by species-specific PCR targeting the sodA gene. Universal ciprofloxacin resistance (100%) and high-level ampicillin resistance (75–80%) were recorded; the genetic basis of fluoroquinolone resistance was not characterised. All isolates remained fully susceptible to linezolid and tigecycline. One E. faecalis isolate from hospital wastewater displayed a phenotypic profile consistent with vancomycin-resistant Enterococcus (VRE); genotypic confirmation by vanA/vanB PCR was not performed within the scope of this study; this isolate is classified as putative-VRE throughout. The four Lactobacillus spp. strains (designated LBAS-01 to LBAS-04, confirmed at genus level by genus-specific PCR) were confirmed free of transferable antibiotic resistance genes and haemolytic activity. All produced measurable in vitro inhibitory activity against MDR enterococci (inhibition zone diameters: 9–20 mm); partial mechanistic characterisation indicated that two strains produce a proteinaceous inhibitory substance consistent with bacteriocin activity.