The use of diphenyl ether (DE) and its 4-monohalogenated derivatives (4-HDE) as flame retardants, solvents, and substrates in biocide production significantly increases the risk of ecosystem contamination. Their removal is important from the point of view of environmental protection. The aim of this study was to evaluate the degradation processes of DE and 4-HDE by enzymes of the environmental bacterial strains under one-substrate and co-metabolic conditions. The study is focused on the biodegradation of DE and 4-HDE, the enzymatic activity of microbial strains, and the cell surface properties after contact with compounds. The results show that the highest biodegradation (96%) was observed for 4-chlorodiphenyl ether in co-metabolic culture with P. fluorescens B01. Moreover, the activity of 1,2-dioxygenase during degradation of 4-monohalogenated diphenyl ethers was higher than that of 2,3-dioxygenase for each strain tested. The presence of a co-substrate provoked changes in dioxygenase activity, resulting in the increased activity of 1,2-dioxygenase. Moreover, the addition of phenol as a co-substrate allowed for increased biodegradation of the diphenyl ethers and noticeable modification of the cell surface hydrophobicity during the process. All observations within the study performed have led to a deeper understanding of the contaminants’ biodegradation processes catalyzed by environmental bacteria.