The aim of this study is to comparative study between conventional and molecular assay of isolation, identification and characterization of non-leguminous endophytic bacteria and fungi in the leguminous root samples. The plant root samples, Helianthus annuus, Carica papaya and Lycoperesicum solanum (Sunflower root and stem, pawpaw root and stem, and tomato root and stem from Adekunle Ajasin University School farm, Akungba Akoko, Ondo state, Nigeria. The isolation of endophytic bacteria were performed using the conventional method of isolation (biochemical test) and characterization were done using both the conventional and molecular method of bacteria characterization. The antibiotic susceptibility test (Antibiogram) was observed using disc diffusion. The four bacteria identified were Bacillus cereus, Enterobacter sp. Actnomycoses sp. and Aeromonas sp. for conventional method and Fusarium solani, Fusarium vortecelium and Bacillus thuringiensis for molecular method as confirmatory point of view. In this study, all isolated organisms tends to be Gram positive using the gram staining technique. Antibiogram shows the zones of inhibition with diameter ranging from 0-20 mm, Enterobacter sp. were more sensitive to the various antibiotics used. Ultraviolet spectrophotometer was also used to determine the growth dynamic as well as the death rate of the isolates, the addition of antibiotics (ciprofloxacin) to the isolates at the 24th hour speed up the death rate of the isolates from non-leguminous endophytic bacteria. After the preliminary identification of the bacteria isolates and the confirmatory identification of both bacteria and fungi isolates of the non-leguminous endophytic microorganism, it was noted that the preliminary identification was only able to achieve the genus level of taxonomic characterization, While the molecular method confirm the molecular sub level identification of isolates depletes the absolute taxonomic identification and characterization to the sub-species level. The results of this study validates the use of molecular sequencing for the assay identification and characterization of non-leguminous endophytic bacteria and fungi as the easy and best mode of identification of both bacteria and fungi isolates as a veritable tools for research purposes.