Cyanobacteria represent promising organic inputs in rice–wheat cropping system, as they contribute towards accretion of N and C, besides secreting growth-promoting substances which influence plant productivity and soil fertility. The present study focused towards using a combinatorial approach for evaluating field-level colonization of cyanobacteria in soil and their effect on soil microbiological and plant parameters, employing agronomic and molecular tools. A consortium of cyanobacterial strains (BF1, Anabaena sp., BF2, Nostoc sp., BF3, Nostoc sp. and BF4, Anabaena sp.) was employed in different three-and four-member combinations along with 75% N + Full dose of P and K fertilizers. A significant enhancement in microbial activity and plant growth/yields and savings of 25% N in the wheat–rice cropping sequence were recorded, especially in treatments involving 75% N + Full dose of PK+BF1+BF2+BF4 and T5, i.e. 75% N + Full dose of PK+BF1+BF2+BF3. Such treatments were significantly higher or statistically at par with fertilizer controls – 75% N + Full dose of PK fertilizers. The use of DNA-based markers further helped to establish the colonization of the inoculated cyanobacteria, especially BF2 and BF3 strains. Our study clearly illustrated the establishment of inoculated cyanobacterial strains and their role in enhancing the crop productivity and soil health of the rice–wheat cropping system.