This study evaluated replacing synthetic nitrogen with organic and bio-fertilizers to improve nutrient efficiency in barley (Hordeum vulgare) grown in sandy calcareous soil. A pot experiment tested varying mineral nitrogen rates against organic sources—compost, chicken manure, and pigeon dung—applied individually or integrated with microbial bio-fertilizers.The results showed that while high mineral nitrogen lowered soil pH to temporarily aid availability, integrating organic manures with bio-fertilizers significantly maximized essential micronutrient uptake, specifically iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu), in both barley straw and grains. Chicken manure combined with bio-fertilizer inoculation yielded the highest structural growth and nutrient accumulation by optimizing rhizosphere conditions and preventing calcareous nutrient locking. Ultimately, the study concludes that combining organic and bio-fertilizers can substitute up to 50% of chemical nitrogen inputs, maintaining high yields while enhancing grain nutritional quality and sustainable soil health.