Streptococcus pneumoniae is a leading cause of infectious disease globally. Nasopharyngeal colonization occurs in biofilms and precedes infection. Prior studies have indicated that biofilm-derived pneumococci are avirulent. However, influenza A virus (IAV) infection releases virulent pneumococci from biofilms in vitro and in vivo. Triggers of dispersal include IAV-induced changes in the nasopharynx, such as increased temperature (fever) and extracellular ATP (tissue damage). We used whole-transcriptome shotgun sequencing (RNA-seq) to compare the S. pneumoniae transcriptome in biofilms, bacteria dispersed from biofilms after exposure to IAV, febrile-range temperature, or ATP, and planktonic cells grown at 37°C. Compared with biofilm bacteria, actively dispersed S. pneumoniae, which were more virulent in invasive disease, upregulated genes involved in carbohydrate metabolism. Enzymatic assays for ATP and lactate production confirmed that dispersed pneumococci exhibited increased metabolism compared to those in biofilms. Dispersed pneumococci also upregulated genes associated with production of bacteriocins and downregulated colonization-associated genes related to competence, fratricide, and the transparent colony phenotype. IAV had the largest impact on the pneumococcal transcriptome. Similar transcriptional differences were also observed when actively dispersed bacteria were compared with avirulent planktonic bacteria. Our data demonstrate complex changes in the pneumococcal transcriptome in response to IAV-induced changes in the environment. Our data suggest that disease is caused by pneumococci that are primed to move to tissue sites with altered nutrient availability and to protect themselves from the nasopharyngeal microflora and host immune response. These data help explain pneumococcal virulence after IAV infection and have important implications for studies of S. pneumoniae pathogenesis.