The aims of the study were to evaluate the effect of combined sewer overflows (CSO) and storm water drains (SWD) on metal bioavailability in small urban streams in Prague and to evaluate levels of metals in water, sediment, and macroinvertebrates. The following working hypotheses were adopted: (a) sites dominantly affected by SWD are less polluted by metals, and (b) sites dominantly affected by SWD have higher bioavailability of metals. Physical and chemical parameters (pH, conductivity, NO3-N, NH4-N, COD, alkalinity, and water hardness) and concentrations of the metals Cu, Zn, Pb, Ni, and Cr were determined in samples of water and sediment at five to six sites on four target streams—Zatisský Creek, Kosikovský Creek, Botic Creek, and Rokytka Creek—three to five times per year during the period 2002–2004. The sites from all studied creeks were categorized into five groups according to the prevailing type of urban drainage impact. Macroinvertebrates were sampled and analyzed for metals at each site for a period of 1 month. The concentration patterns of metals were interpreted by partition coefficient (Kd), hazard quotient (HQ), cumulative criterion unit (CCU), and biota sediment accumulation factor (BSAF). Concentrations of metals in water as well as in sediment at sites receiving water from SWD were lower than at sites where creeks receive water from CSO, except for Pb. Concentrations of Cu, Zn, and Pb in sediment were higher at sites affected by CSO. Concentrations of metals in aquatic macroinvertebrates, expressed as BSAF, indicated higher values at sites affected by SWD. Frequencies of high BSAF (>1) were lower in CSO compared to SWD. This finding was explained by (a) a decrease of pH on SWD sites compared to the increase of pH on CSO sites during rain events, (b) a greater resuspension of sediment at SWD sites during rain events, and (c) an abundance of organic matter in CSO available for sorption of metals and a corresponding reduction of their bioavailability. In the study area, the type of urban drainage affects the bioavailability of metals—while SWD increase metal bioavailability, CSO cause its decrease. The sediments in SWD sites do not indicate risk to the benthic community according to the applied environmental quality standards. Water and sediment in creeks affected by SWD are less polluted by metals. Both working hypotheses were therefore supported.