The relentless spread of antimicrobial-resistant Escherichia coli underscores the critical need for alternative therapeutics, such as bacteriophages. A major translational hurdle is the efficient and safe initial isolation of lytic phages with defined therapeutic potentials. This study established and validated a safety-conscious pipeline using the non-pathogenic laboratory strain E. coli DH5α (BSL-1) for environmental phage screening, a strategy designed for accessibility and to circumvent early biosafety concerns. After continuous screening, we isolated a novel lytic phage, vB_EcoS_NLE251, from the soil. Comprehensive phenotyping revealed robust stability (tolerant up to 60 °C) and efficient replication, with an optimal multiplicity of infection (MOI) of 100, yielding a high titer of 2.1 × 10¹⁰ PFU/mL. Crucially, host-range determination using a panel of 35 clinical E. coli isolates showed that NLE251 only exhibited lytic activity in spot assays against a single biliary tract infection-derived strain (54959124), though subsequent quantitative infection efficiency data indicate extremely low productive infection against this target strain. Genomic analysis (49,336 bp dsDNA, belonging to the class Caudoviricetes) further confirmed the absence of virulence or antibiotic resistance genes, fulfilling a key safety prerequisite for therapeutic use. Although NLE251 is a narrow-spectrum agent, this study provides a validated methodological framework that prioritizes safety and specificity from the initial isolation step, offering a safety-prioritized, standardized pipeline for accelerating phage discovery against multidrug-resistant bacterial infections.