Abstract Objectives Bacterial biofilms developing in the bronchial tree of patients experiencing acute exacerbations of chronic bronchitis (AECBs) are suggested to cause relapses and recurrences of the disease because the matrix barrier impairs antibiotic access to the offending organisms. We examined whether bronchodilators could modulate pneumococcal biofilm development and antibiotic action using an in vitro model. Methods Streptococcus pneumoniae strains from patients hospitalized for AECBs and two reference strains (ATCC 49619 and R6) were screened for biofilm formation (multi-well plates; 2–11 days of growth). Ipratropium and salbutamol (alone or in combination) were added at concentrations of 1.45 and 7.25 mg/L, respectively (mimicking those in the bronchial tree), and their effects were measured on biofilm formation and modulation of the activity of antibiotics [full antibiotic concentration-dependent effects (pharmacodynamic model)] with a focus on moxifloxacin and solithromycin. Bacterial viability and biomass were measured by the reduction of resazurin and crystal violet staining, respectively. Release of sialic acid (from biofilm) and neuraminidase activity were measured using enzymatic and HPLC–MS detection of sialic acid. Results All clinical isolates produced biofilms, but with fast disassembly if from patients who had received muscarinic antagonists. Ipratropium caused: (i) reduced biomass formation and faster biofilm disassembly with free sialic acid release; and (ii) a marked improvement of antibiotic activity (bacterial killing and biomass reduction). Salbutamol stimulated neuraminidase activity associated with improved antibiotic killing activity (reversed by zanamivir) but modest biomass reduction. Conclusions Ipratropium and, to a lesser extent, salbutamol may cooperate with antibiotics for bacterial clearance and disassembly of pneumococcal biofilms.