To demonstrate elimination of bacterial biofilm on membranes to represent wastewater treatment as well as biofilm formed by antibiotic‐resistant bacterial (ARB) to signify medical application, an antibiotic‐resistant bacterium and its lytic bacteriophage were isolated from a full‐scale wastewater treatment plant. Based on gram staining and complete 16 S rDNA sequencing, the isolated bacterium showed a more than 99% homology with Delftia tsuruhatensis, a gram‐negative bacterium belonging to β‐proteobacteria. The Delftia lytic phage's draft genome revealed the phage to be an N4‐like phage with 59.7% G + C content. No transfer RNAs were detected for the phage suggesting that the phage is highly adapted to its host Delftia tsuruhatensis ARB‐1 with regard to codon usage, and does not require additional tRNAs of its own. The gene annotation of the Delftia lytic phage found three different components of RNA polymerase (RNAP) in the genome, which is a typical characteristic of N4‐like phages. The lytic phage specific to D. tsuruhatensis ARB‐1 could successfully remove the biofilm formed by it on a glass slide. The water flux through the membrane of a prototype lab‐scale membrane bioreactor decreased from 47 L/h m² to ∼15 L/h m² over 4 days due to a biofilm formed by D. tsuruhatensis ARB‐1. However, the flux increased to 70% of the original after the lytic phage application. Overall, this research demonstrated phage therapy's great potential to solve the problem of membrane biofouling, as well as the problems posed by pathogenic biofilms in external wounds and on medical instruments. Biotechnol. Bioeng. 2015;112: 1644–1654. © 2015 Wiley Periodicals, Inc.