The study was aimed to assess the potency of nitrogen (N)-fixing purple non-sulfur bacteria (NF-PNSB) on ameliorating soil quality, N uptake, and yield of pineapple in acid sulfate soil. The experiment, following a completely randomized block design, had two factors: (i) supplementation of biofertilizers containing the NF-PNSB (No applied bacteria, Rhodobacter sphaeroides W15, W39, and a mixture of both W15 and W39) and (ii) levels of N fertilizer [0, 50, 75, and 100% N of recommended fertilizer formula (RFF)]. Results demonstrated that supplying biofertilizers containing the mixture of both W15 and W39 increased the values of pH_H2O and NH₄⁺, and decreased the values of EC, Al³⁺, and Fe²⁺ at a depth of 0–20 cm, in comparison with the no supplied biofertilizers, by 15.8, 39.9, 26.9, 17.8, and 52.9%, respectively. Applying biofertilizers enhanced the total N uptake by 28.9%, resulting in greater growth and a 22.9% increase in pineapple yield, in comparison with the no biofertilizers supplied treatments. Supplying the two strains W15 and W39 not only contributed to reducing 25% N of RFF but also preserved the pineapple yield, compared to the fertilizing 100% N of RFF. Both W15 and W39 are potent for use as crop yield enhancers.