The tomato leaf miner, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is a devastating pest worldwide affecting tomato production. Entomopathogenic nematodes (EPNs) are well known for their potential as biocontrol agents to control the emergence and development of this insect pest. Virulence and pathogenicity of five EPN strains isolated from different fields in Morocco were evaluated against the larval stage of T. absoluta. In laboratory assays, Steinernema feltiae strains (SF-MOR9 and SF-MOR10) showed significantly higher infectivity rates after 72 h compared to Heterorhabditis strains. In leaf bioassay, S. feltiae strains alongside H. bacteriophora(HB-MOR8) strain caused the highest larval mortality rate (80–100%) at 40–50 infective juveniles (IJs) cm−2 which confirms the importance of the dose applied. On top of that, EPNs were able to locate and accurately kill the insect larvae inside and outside leaf conditions. Subsequently, the results showed that both S. feltiae strains were significantly effective in pot experiments for both applied concentrations (40 and 50 IJs cm−2). In addition, the efficiency of these nematodes was assessed under field conditions. Both S. feltiae accessions had optimal effects against T. absolutalarvae with more than 80% mortality rate at 50 IJs cm−2. Heterorhabditis strain (HB-MOR8) significantly reduced larval occurrence with a more than 60% mortality rate when applied at the same dose. Therefore, the three tested indigenous EPN strains; SF-MOR9, SF-MOR10, and HB-MOR8 could be used as promising eco-friendly biological agents against T. absoluta in a broad agronomic range.