Biofilms are more resistant to antibiotics and antimicrobial stressors than planktonic bacteria; however, only a limited number of standardized assays enable investigation of this phenomenon. Here, we utilized non-invasive and independent techniques, including isothermal microcalorimetry (IMC) and tunable diode laser absorption spectroscopy (TDLAS), to measure the effect of isoniazid on metabolic activity and respiratory capability of mature Mycobacterium tuberculosis H37Ra (an avirulent strain) and Mycobacterium smegmatis biofilms. We detected only minor changes in metabolic heat production and respiratory rates (O₂ and CO₂) for mature M. smegmatis biofilms after antibiotic exposure. However, mature M. tuberculosis biofilms showed greater sensitivity to antibiotic treatment, with isoniazid exhibiting dose-dependent effects on metabolic activity and respiration. Specifically, treatment of M. tuberculosis biofilms with 250 μg/ml and 1 mg/ml isoniazid decreased the rate of heat production by 33% and 40%, respectively, oxygen consumption by 18% and 55%, respectively, and carbon dioxide production by 27% and 64%, respectively. These effects were prominent even after regrowth of antibiotic-treated M. tuberculosis H37Ra biofilms on fresh medium. Our data therefore suggest that IMC and TDLAS are appropriate for drug susceptibility testing of mature biofilms, and these techniques may facilitate study of microbial resistance to antimicrobial compounds from a bioenergetic perspective.