Plant growth-promoting rhizobacteria (PGPR) boosts agricultural productivity and alleviates environmental stresses by forming biofilms under natural climatic conditions. In the past few years, microorganisms in biofilm have gained impetus for efficient root colonization. The current work aims to characterize biofilm-associated rhizobacteria for wheat growth and yield enhancement. In this study native rhizobacteria were isolated from the wheat rhizosphere and ten isolates were characterized for plant growth promoting traits and biofilm production under axenic conditions. Among these ten isolates, five potential biofilm-producing plant growth-promoting rhizobacteria on the basis of invitro plant growth promoting trait assays were further tested under controlled and field conditions on wheat growth and yield attributes. Surface-enhanced Raman spectroscopy (SERS) spectra further revealed that biochemical contents of biofilm produced by selected bacterial PGPR strains are associated with proteins, carbohydrates, lipids, amino acids and DNA/RNA. Inoculated plants in growth chamber resulted in longer roots, shoots, and increase in fresh biomass than controls. Similarly, significant increases in plant height (up to 13.3, 16.7%), grain yield (up to 29.6, 46.9%), number of tillers (up to 18.7, 34.8%), nitrogen contents (up to 58.8, 48.1%), and phosphorus contents (up to 63.0, 51.0%) in grains were seen in both pot and field trials, respectively. The two most promising biofilm-producing isolates were identified through 16s rRNA partial gene sequencing as Brucella sp. (BF10), Lysinibacillus macroides (BF15). Moreover, leaf pigmentation and relative water contents increased in all treated plants. Taken together, our results revealed that biofilm forming PGPR can boost crop productivity for sustainable agriculture.