The present study reports the use of Citrus limetta (CL) residue for cultivating Chlorella sp. mixotrophically to augment production of biodiesel. The cultivation of Chlorella sp. using CL as media was carried out by employing a fed-batch technique in open tray (open tray+CL) and in software (BioXpert V2)-attached automated photobioreactor (PBR+CL) systems. Data showed the limit of nitrogen substituent and satisfactory organic source of carbon (OSC) in CL, causing > 2-fold higher lipid content in cells, cultivated in both the systems than in control. For the cells grown in both the systems, ≥ 3-fold enhancement in lipid productivity was observed than in control. The total fatty acid methyl ester (FAME) concentrations from lipids extracted from cells grew in PBR+CL and in open tray+CL techniques were calculated as 50.59% and 38.31%, respectively. The PBR+CL system showed improved outcomes for lipid content, lipid and biomass productivity, FAME characteristics and physical property parameters of biodiesel than those obtained from the open tray+CL system. The physical property parameters of biodiesel produced from algal cells grown in PBR+CL were comparable to existing fuel standards. The results have shown lower cold filter plugging point (- 6.57 °C), higher cetane number (58.04) and average oxidative stability (3.60 h). Collectively, this investigation unveils the novel deployment of CL as a cost-effective feedstock for commercialisation of biodiesel production.