| Date: | 31 October – 6 November |
| Edition: | 12th |
| Surface: | Clay |
| Location: | Guayaquil, Ecuador |
| Champs: |  Nicolás Kicker |
| Champd: | Ariel Behar /  Fabiano de Paula |
The 2016 Challenger Ciudad de Guayaquil was a professional tennis tournament played on clay courts. It was the twelfth edition of the tournament which was part of the 2016 ATP Challenger Tour. It took place in Guayaquil, Ecuador, between October 31 and November 6, 2016.
| Country | Player | Rank1 | Seed | |
|---|---|---|---|---|
| 57 | 1 | |||
| Argentina | 67 | 2 | ||
| Argentina | ||||
| Argentina | Carlos Berlocq | 89 | 4 | |
 Dominican Republic | 94 | 5 | ||
 Colombia | Santiago Giraldo | 100 | 6 | |
| Brazil | Rogério Dutra Silva | 105 | 7 | |
| Brazil | João Souza | 125 | 8 | |
| Ernesto Escobedo | 126 | 9 |
The following players received wildcards into the singles main draw:
The following players received entry from the qualifying draw:
Daniel Elahi Galán Bastián Malla Andrés Molteni João Pedro SorgiThe following player entered as a lucky loser:
See main article: 2016 Challenger Ciudad de Guayaquil – Singles.
Nicolás Kicker def. 
Arthur De Greef 6–3, 6–2See main article: 2016 Challenger Ciudad de Guayaquil – Doubles.
Fabiano de Paula def. Marcelo Arévalo / 
Sergio Galdós 6–2, 6–4
