There is a continuing argument about the benefits of biochar on arbuscular mycorrhizal (AM) symbiosis, crop growth, yield, and fertility of soil. There is also limited research on the effects of biochar on AM colonization, cucumber yield, and soil fertility improvement. Therefore, this investigation aimed to determine the impact of poultry litter biochar (PLB) on colonization of roots by indigenous AM fungi in agricultural soil and their contribution to cucumber yield, nutrition, and soil fertility improvement. A field trial was conducted to assess the effect of PLB combined with compound poultry manure (CPM) and nitrophos (NP) fertilizer to investigate the response of treatments on nutrient-deficient sandy soils. Plant growth responses to biochar showed better plant growth and yield of cucumber. Application of biochar with and without CPM and NP reduced the negative impact of nutrient deficiency stress on cucumber growth. AM fungal colonization, soil fertility, and cucumber yield were improved with the combined application of biochar, CPM, and NP fertilizer. Post-harvest, soil C, N, P, K, Ca, Mg, S, Zn, Cu, Fe, and Mn increased with application of biochar applied with CPM and NP. Biochar application with CPM and NP also increased the percent root colonization of cucumber. Use of biochar with CPM and NP has the potential to improve plant growth, yield, nutrient uptake, and soil fertility. Further studies in various agro-ecological conditions would help utilize this technology in sustainable crop production.