The scope of the present study is the estimation of key operational parameters of a drinking water treatment plant (DWTP), particularly the dosages of treatment chemicals, using artificial neural networks (ANNs) based on measurable in situ data. The case study consists of the Aposelemis DWTP, where the plant operator had an estimation of the ANN output parameters for the required dosages of water treatment chemicals based on observed water quality and other operational parameters at the time. The estimated DWTP main operational parameters included residual ozone (O3) and dosages of the chemicals used: anionic polyelectrolyte (ANPE), poly-aluminum chloride hydroxide sulfate (PACl), and chlorine gas (Cl2(g)). Daily measurable results of water sample analysis and recordings from the DWTP Supervisory Control and Data Acquisition System (SCADA), covering a period of 38 months, were used as input parameters for the artificial neural network (1188 values for each of the 14 measurable parameters). These input parameters included: raw water supply (Q), raw water turbidity (T1), treated water turbidity (T2), treated water residual free chlorine (Cl2), treated water concentration of residual aluminum (Al), filtration bed inlet water turbidity (T3), daily difference in water height in reservoir (∆H), raw water pH (pH1), treated water pH (pH2), and daily consumption of DWTP electricity (El). Output/target parameters were: residual O3 after ozonation (O3), anionic polyelectrolyte (ANPE), poly-aluminum chloride hydroxide sulfate (PACl), and chlorine gas supply (Cl2(g)). A total of 304 different ANN models were tested, based on the best test performance (tperf) indicator. The one with the optimum performance indicator was selected. The scenario finally chosen was the one with 100 neural networks, 100 nodes, 42 hidden nodes, 10 inputs, and 4 outputs. This ANN model achieved excellent simulation results based on the best testing performance indicator, which suggests that ANNs are potentially useful tools for the prediction of a DWTP’s main operational parameters. Further research could explore the prediction of water chemicals used in a DWTP by using ANNs with a smaller number of operational parameters to ensure greater flexibility, without prohibitively reducing the reliability of the prediction model. This could prove useful in cases with a much higher sample size, given the data-demanding nature of ANNs.