The agriculture sector has circumpassed a massive revolution in the past few decades. Old conventional practices and modern innovative techniques have complemented each other well. Applications of fertilizers have increased the crop yield, but its higher concentration has offered detrimental effects. Farmers often exceed the prerequisite limit and this exaggeration denatures the soil and this aggravates various ecological repercussions. To tackle this complication, organic bio fertilizers have emerged as a sustainable, eco-friendly substitute and cost-effective approach. The objective of the present experimental setup is to investigate the effects of fertilizer ammonium Sulphate and biofertilizer agrozyme at the cytological level in the root meristems of barley and to trace out a safer crop enhancer between the two. Various concentrations of fertilizer and biofertilizer used were 50%, 100%, 150%, 200% each, along with a control set. Chromosomal studies revealed a considerable impact on Active mitotic index which depicted a consistent increment in the case of bio fertilizer, but appeared to decline at higher doses in the case of fertilizer, with respect to the control. Various chromosomal anomalies were observed, such as scattering, precocious movement, stickiness, fragmentation at metaphase and stickiness, laggard, unorientation, bridges etc. at anaphase. Percentage of these abnormalities was higher in the case of fertilizer. Conclusively, it is inferred that Bio fertilizers are mitotically accelerating and chromotoxically much safer as compared to fertilizers and should be promoted for agro-system to obtain sustainable crop improvement.