Tomato (Lycopersicon esculentum Mill.) is a soft fruit and has a very short shelf life. Environmental factors decreased its quality and shelf life like microbial diseases and high respiration and transpiration rate. Biopreservation is a novel technique for the preservation of food products by the utilization of natural antimicrobials substances to increase the shelf life of fruits and vegetables. Fruit and vegetables are naturally soft and they want protection against microorganisms during formulation, preservation, and supply. The objective of this research was to evaluate the effect of bio-preservatives such as Turmeric, Aloe vera and Moringa on the shelf life of tomato fruit at room temperature. Treatments were applied in a completely randomized design (CRD) with three replications. Bio-preservatives aqueous solutions with 10% (w/v) concentration were used. All morphological, physiochemical, and biochemical analysis were done after every 5 days. Highest weight loss and decrease in firmness were observed in control set and lowest in T4 (Turmeric + Aloe vera 10%) coated tomatoes. Similarly, the highest reduction in pH and TA were observed in the control set and lowest in T4. The cellulase enzyme activity increased with the storage time. T4 (Turmeric + Aloe vera 10%) showed minimum increase in cellulase activity. Thus, it is concluded that T4 showed significant results and thus caused an increase in the shelf life of tomato fruit. It is also concluded that Bio-preservatives such as Turmeric, Aloe vera, Moringa, are recommended to enhance the shelf life and physiochemical constituents of tomato (Lycopersicon esculentum Mill.) fruit at room temperature. Biopreservatives are easily available, low cost, ecofriendly and have no health hazards as compared to chemical preservatives.