This paper discusses the microbial basis and the latest research on nitrous oxide (NO) emissions from biofilms processes for wastewater treatment. Conditions that generally promote NO formation in biofilms include (1) low DO values, or spatial DO transitions from high to low within the biofilm; (2) DO fluctuations within biofilm due to varying bulk DO concentrations or varying substrate concentrations; (3) conditions with high reaction rates, which lead to greater formation of intermediates, e.g., hydroxylamine (NHOH) and nitrite (NO), that promote NO formation; and (4) electron donor limitation for denitrification. Formation of NO directly results from the activities of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and heterotrophic denitrifying bacteria. More research is needed on the roles of AOA, comammox, and specialized denitrifying microorganisms. In nitrifying biofilms, higher bulk ammonia (NH) concentrations, higher nitrite (NO) concentrations, lower dissolved oxygen (DO), and greater biofilm thicknesses result in higher NO emissions. In denitrifying biofilms, NO accumulates at low levels as an intermediate and at higher levels at the oxic/anoxic transition regions of the biofilms and where COD becomes limiting. NO formed in the outer regions can be consumed in the inner regions if COD penetrates sufficiently. In membrane-aerated biofilms, where nitrification takes place in the inner, aerobic biofilm region, the exterior anoxic biofilm can serve as a NO sink. Reactors that include variable aeration or air scouring, such as denitrifying filters, trickling filters, or rotating biological contactors (RBCs), can form peaks of NO emissions during or following a scouring or aeration event. NO emissions from biofilm processes depend on the microbial composition, biofilm thickness, substrate concentrations and variability, and reactor type and operation. Given the complexity and difficulty in quantifying many of these factors, it may be difficult to accurately predict emissions for full-scale treatment plants. However, a better understanding of the mechanisms and the impacts of process configurations can help minimize NO emission from biofilm processes for wastewater treatment.