Antibiotic resistance can occur via three general mechanisms: prevention of interaction of the drug with target, efflux of the antibiotic from the cell, and direct destruction or modification of the compound. This review discusses the latter mechanisms focusing on the chemical strategy of antibiotic inactivation; these include hydrolysis, group transfer, and redox mechanisms. While hydrolysis is especially important clinically, particularly as applied to beta-lactam antibiotics, the group transfer approaches are the most diverse and include the modification by acyltransfer, phosphorylation, glycosylation, nucleotidylation, ribosylation, and thiol transfer. A unique feature of enzymes that physically modify antibiotics is that these mechanisms alone actively reduce the concentration of drugs in the local environment; therefore, they present a unique challenge to researchers and clinicians considering new approaches to anti-infective therapy. This review will present the current status of knowledge of these aspects of antibiotic resistance and discuss how a thorough understanding of resistance enzyme molecular mechanism, three-dimensional structure, and evolution can be leveraged in combating resistance.